Phosphorus fertilizer bio-catalyst for sustainable crop production

ABSTRACT

Endophytic microbial strains as biocatalysts isolated from fresh plant samples, compositions, and methods of use thereof to enhance the growth and/or yield of a plant in the presence of reduced synthetic phosphate fertilizers are provided. Endophytic microbial strains serve as biocatalysts to solubilize mineral-P and mineralize organic-P otherwise unavailable to plants for their nutritional phosphate needs. Thus defined, biocatalysts, will serve to replace synthetic phosphate fertilizers. Also provided are materials and methods for inoculating plants with these biocatalysts at carefully selected inoculum densities to reliably reduce the amount of synthetic phosphate fertilizer by 50% thus accomplishing obtaining optimal yields in technically and cost-effective manner.

FIELD OF THE INVENTION

The present invention relates to the field of sustainable agriculture.Specifically, the disclosure provides novel purified bacterialpopulations comprising plant isolated endophytes and syntheticcombinations of seeds with heterologously plant derived endophytes toprovide a specific benefit that of optimal production of crop plants inthe absence of synthetic phosphate fertilizers or in the presence ofsynthetic phosphate fertilizers applied at the rate of 30-50% less thanthe recommended phosphorus fertilizer application rate. In particular,the synthetic compositions and methods disclosed herein are useful forenhancing plant growth in the complete absence of synthetic phosphatefertilizers or in their much reduced presence.

BACKGROUND OF THE INVENTION

The United States is, by far, the largest producer of corn in the world,producing as much as 35% of world's corn, 33% of world's soybean, morethan 65% of world's sorghum and is the biggest wheat exporter (Ag 101,US EPA).

Although US currently leads the world in corn production, the portion ofagricultural revenue returned to farmers decreased from 37% to 19% from1950 to 2002 (Atwell et at, 20111). In addition, input costs increasedsevenfold and the real price of corn adjusted for inflation decreasedfivefold (Duffy, 206).

One major contributor to the input costs is the cost of fertilizer andpesticides because US farming is fertilizer and pesticide intensive.

The cost of phosphate fertilizer has risen steadily from the year, 2000doubling in the year 2007 (Olcyzk et al., 2007).

Phosphorus fertilizer manufactured from phosphorus rock is mined in theUS (Florida), China, Morocco, and Russia. In year 2006 alone, 142million tons was mined worldwide (Olcyzk et al. 2007), yet, at thecurrent world-wide rate of application of fertilizers, the readilyavailable sources of high grade phosphate rocks will be depleted withinthe next 60 to 90 years (Runge-Metzger, 1995).

The situation is critical because the supply of phosphate rock inFlorida may be exhausted as early as year 2040 according to theInstitute of Phosphate Research (Olcyzk et al., 2007).

Phosphate fertilizers have been critical to crop growth becausephosphorus deficiency often limits plant growth (Schachtman, et al.1998; Vance, et al., 2003; Raghothama, and Karthikeyan, 2005) althoughthis essential plant macronutrient is critically important for improvingsoil fertility in both tropical and temperate regions (Von Uexkûll &Mutert. 1995).

The practice of applying chemical (inorganic) P fertilizers to alleviateP deficiency is inefficient for both logistical and economic reasonshowever, because 75-90% of soluble P from fertilizers rapidly becomesimmobilized as Fe-, Al-, or Ca—PO₄ phases in soils (Gyaneshwar et al.,2002).

Thus effective means for utilizing residual accumulated insoluble P insoils and a means for increasing efficiency of applied synthetic Pfertilizers are critically needed.

This takes on particular significance when we note that global foodproduction needs to increase by 50% in the next 20 years to sustain theincreasing world population and prosperity.

Although theoretical estimates have suggested that the accumulated P inagricultural soils is sufficient to sustain maximum crop yieldsworldwide for about 100 years (Gyaneshwar et al., 202), most soils aredeficient in bioavailable P.

Therefore, sustainable alternatives to improving P bioavailability areneeded for maintaining U.S.'s edge in crop production and agriculturalproductivity in general.

The proposed embodiment will address the current need for sustainableand cost efficient P management in soils by developing a biocatalystthat makes mineral soil-P, and organic-P available for plant needs thusreducing or eliminating the need for the application of synthetic Pfertilizers by a combination of utilization of already available soil-Pand by increasing the efficiency of applied synthetic-P fertilizer.

The proposed embodiment will have the additional benefit of improvingsurface and ground water quality because application of manures andfertilizers has resulted in increased transfer of soil P (solidassociated) to solution and eventually, via erosion and runoff, tosurface waters where it plays a key role in eutrophication andimpairment of affected waters as a resource for drinking, recreation andindustry.

The total soil P content typically varies between 500 and 2000 mg kg⁻¹(Vance et al., 20113).

Of this, typically 30-50% of the total insoluble P is present as organicP (P_(o)) mainly as inositol phosphate and the remaining is found asFe-, Al- or Ca-associated mineral phosphate (P_(i)); phosphate dissolvedin soil solution ranges between only 0.1 and 10 μM (Bielski, 1973;Ozanne, 1980; Raghothama, 1999; Frossard et al., 2000).

Because plants can only take up phosphorus as dissolved H_(x)PO₄ ^(y-)ions, and for optimal crop growth 0.5-0.7 mM dissolved P is needed inthe soil solution, most soils are P deficient and crop growth is oftenlimited by P bioavailability.

Application of chemical fertilizers i.e., phosphate salts is used tosupplement the limited pools of dissolved P; however, because of highaffinity of P binding to Fe-oxyhydroxide. Al-oxyhydoxide minerals andprecipitation as calcium-phosphate phases, dissolved P is quicklyconverted to insoluble P.

The main goal of the preferred embodiment then is to increase theefficacy of chemical fertilizers and to use the already existingsubstantial reserves of insoluble soil-P, thereby reducing theapplication of chemical fertilizers.

Toward this goal, we enhance dominant natural mechanisms of insolublesoil-P bioavailability.

Two main mechanisms of making Pi and Po bioavailable for plant'snutritional needs involves secretion of organic acids to solubilize Piand secretion of phosphatase enzymes for mineralization of Po. Theseorganic acids and phosphatase enzymes are secreted both by soilmicroorganisms (bacteria and fungi) and to a smaller extent by plantroots in response to P deficiency (Raghothama and Karthikeyan, 2005;Martinez, 1967).

Although both bacteria, and fungi are ubiquitous in soils, Psolubilizing and Po mineralizing bacteria (phosphobacteria) generallyoutnumber their fungal counterparts by 2-150 fold (Hilda and Fraga,1999); the P solubilization potential of phosphobacteria can thereforebe enhanced to serve as an effective biocatalyst in making insoluble‘fixed’ P plant available in an eco-friendly, reliable and sustainablemanner.

Phosphobacteria have both epiphytic and endophytic modes of associationwith the host plant (corn, soybean, wheat, and sorghum, and other cropsby extension) and the mode of association can affect the efficacy ofphosphobacteria.

Colonizing the plant root epiphytically is difficult because theinoculant has to compete with the native soil bacteria (Kozyrovska etal., 1996).

Phosphobacteria with endophytic relationship with host plant residewithin apoplastic spaces inside the host plant thus keeping them awayfrom the natural biocenosis giving them a significant edge in competingwith the soil bacteria (Kozyrovska et al. 0.1996; Sturz et al., 20011).

Because, endophytes live within the plant, they can recover more easilyfrom stress situation; they may also form beneficial host-endophyteallelopathies thus protecting the plant from superinfection by soilbacteria (Kozyrovska et al., 1996; Sturz et al., 2010).

SUMMARY OF THE INVENTION

The proposed embodiment pertains to the development of biofertilizerconsisting of endophytic phosphobacteria (BioCat-P) inoculated cropseeds as an environmentally sustainable, and cost-effective alternativeto synthetic fertilizers and/or applied in combination with thesynthetic-P fertilizers as a means of increasing the efficacy ofsynthetic fertilizers. The present invention relates to the field ofsustainable agriculture. Specifically, the disclosure provides microbialcompositions and methods useful for the optimal production of cropplants in the absence of synthetic phosphate fertilizers or in thepresence of synthetic phosphate fertilizers applied at the rate of30-50% less than needed. In particular, the compositions and methodsdisclosed herein are useful for enhancing plant growth in the completeabsence of synthetic phosphate fertilizers or in their much reducedpresence.

LIST OF REFERENCES

-   Atwell R. C., Schulte L. A., Westphal L. M., How to build    multifunctional agricultural landscapes in the US Corn Belt: Add    perennials and partnerships, Land Use Policy, 27 (2010) 1082-1090.-   Bieleski, R. L., Phosphate pools, phosphate transport, and phosphate    availability, Ann. Rev. Plant Physiol. 24 (1973) 225-252.-   Frossard E., Condron L. M., Oberson A., Sinaj S., and Fardeau J. C.,    Processes governing phosphorus availability in temperate soils., J.    Environ. Qual. 29 (2000), 12-53.-   Gyaneshwar, P., Kumar, G. N., Parekh, L. J., and Poole, P. S., Role    of soil microorganisms in improving P nutrition of plants. Plant and    Soil 245 (2002) 83-93.-   Hilda R. and Fraga R., Phosphate solubilising bacteria and their    role in plant growth promotion. Biotechnology Advances 17 (1999)    319-339.-   Kozyrovska, N., Kovtunovych, G., and Groosova, E. Kuharchuk, P., and    Kordyum, V., Novel inoculants for an environmentally-friendly crop    production. Resources Conservation and Recycling, 18 (1996) 79-85.-   Martinez, J. R., Organic phosphorus mineralization and phosphatase    activity, Folia Microbiologica, 13 (1967) 161-&-   Olczyk, T., Yuncong, L., Edward, E., Na-Lampag, S., and Fan,    X., 2007. Updates on Fertilizer prices. University of Florida IFAS.-   Ozanne P. G., 1980 Phosphate nutrition of plants—general treatise.    In The role of phosphorus in agriculture. Eds. F E Khasawneh, E C    Sample and E J Kamprath. pp. 559-589. American Society of Agronomy,    Crop Science Society of America, Soil Science Society of America,    Madison, Wis., USA.-   Raghothama K. G., Phosphate acquisition. Ann. Rev. Plant Physiol.    Mol. Biol. 50 (1999) 665-693.-   Raghothama, K. G. and Karthikeyan, A. S. Phosphate acquisition,    Plant Soil 274 (2005) 37-49.-   Runge-Metzger, A., 1995, Closing the cycle: Obstacles to efficient P    management for improved global food security. In Phosphorus in the    Global Environment: Transfers, cycles and Management. Ed. H Tiessen.    pp. 27-42. John Wiley and Sons, NY.-   Schachtman, D. P., Reid, R. J., Ayling, S. M., Phosphorus uptake by    plants from soil to cell, Plant Physiol. 116 (1998) 447-453.-   Sturz, A. V, Christie, B. R., Nowak, J., Bacterial endophytes:    Potential role in developing sustainable systems of crop production,    Critical reviews in plant sciences, 19 (2000) 1-30.-   Vance, C. P., Uhde-Stone, C., Allan, D. L., Phosphorus acquisition    and use: critical adaptations by plants for securing a nonrenewable    resource, New Phytol. 157 (2003) 423-447.-   Von Uexküll H. R., Mutert, E., 1995. Global extent, development and    economic-impact of acid soils. Plant and soil 171 (1995) 1-15.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As part of our invention, we isolated and purified a number ofendophytic bacteria from corn, sorghum and sugarcane plant parts. Allthe isolated and purified bacterial strains were screened quantitativelyfor their ability to function as endophytic phosphobacteria. This wasaccomplished by screening for production of organic acids forsolubilizing inorganic/mineral phosphorus; and for the production ofacid and alkaline phosphatase enzymes for conversion of organicphosphorus to inorganic phosphorus. In addition, phosphobacteria weretested for heterologous inoculation of corn and sorghum seeds.

We were able to purify 12 bacterial strains of endophyticphosphobacteria from corn, sorghum, and sugarcane plants of which onlyone strain T6 showed production of all three extracellular enzymes forphosphorus solubilization that include organic acids, acid phosphatase,and alkaline phosphatase enzymes. This strain has been deposited asBMS-201 with the NRRL and has the accession number B-67827.

The preferred embodiment comprising synthetic combination of thepurified bacterial strain T6 (NRRL accession number B-67827) when testedfor its heterologous inoculation of corn and sorghum seeds showedexcellent inoculation ability without harming the plant. The preferredembodiment comprising synthetic combination of the purified bacterialstrain T6 heterologously inoculated corn and sorghum seeds wereevaluated in crop systems in hydroponic, simulated soil systems, andreal soils.

The preferred embodiment has included isolation of Pi solubilizing andacid- and alkaline-phosphatase producing endophytic bacteria from plantsamples of corn, sorghum, and sugarcane.

Further, the preferred embodiment has involved determining inoculationefficacy of the isolated endophytic bacteria by determining inoculumdensity in root and shoot of corn and root and shoot of sorghum.

Further, the preferred embodiment has involved using phosphatedeficiency symptoms, root/shoot ratio, and chlorophyll contentmeasurements in demonstrating that corn and sorghum seeds inoculatedwith selected Pi solubilizing endophytic bacteria grown with insolublemineral-P species present in soils such as rock-phosphate andiron-phosphate performed as well or better than non-inoculated controlseeds grown with full strength Hoagland solution containing dissolved P.

FIG. 8 shows healthy and better growth with no soluble/dissolvedphosphorus fertilizer in T6 (NRRL accession number B-67827)heterologously inoculated corn seed compared to the control plants(reference plants that are uninoculated) grown with recommended amountsof phosphorus fertilizer. Uninoculated phosphorus deficient plants incomparison show very poor growth.

In FIG. 9, the ˜40% reduction in root and shoot lengths in the referenceplant in the absence of dissolved phosphorus fertilizer (treatmentlabeled 1) compared to the reference plant grown with recommendedamounts of phosphorus fertilizer (treatment labeled 2) are clearlyindicative of the need for dissolved phosphorus fertilizer for optimalplant growth. In FIG. 9, the plant grown from heterologously inoculatedcorn seed grown in complete absence of any soluble phosphorus fertilizer(see treatments labeled 3 and 4) grew as well or better than thereference plant grown from uninoculated seed with recommended amounts ofdissolved phosphorus fertilizer.

FIG. 10 further shows that the plants grown from T6 (NRRL accessionnumber B-67827) heterologously inoculated corn seed in the absence ofall dissolved phosphorus fertilizer have the best chlorophyll contentswhen compared to uninoculated reference plant grown with recommendedamounts of dissolved phosphorus fertilizer and also surprisingly whencompared to T4 heterologously inoculated corn seed in the absence of alldissolved phosphorus fertilizer.

FIG. 11 shows healthy and better growth with no soluble/dissolvedphosphorus fertilizer in T6 (NRRL accession number B-67827)heterologously inoculated sorghum seed compared to the control plants(reference plants that are uninoculated) grown with recommended amountsof phosphorus fertilizer. Uninoculated phosphorus deficient plants incomparison show very poor growth.

In FIG. 12, the ˜40% reduction in root length and ˜70% reduction inshoot lengths in the reference plant in the absence of dissolvedphosphorus fertilizer (treatment labeled 1) compared to the referenceplant grown with recommended amounts of phosphorus fertilizer (treatmentlabeled 2) are clearly indicative of the need for dissolved phosphorusfertilizer for optimal plant growth. In FIG. 12, plant grown fromheterologously inoculated sorghum seed grown in complete absence of anysoluble phosphorus fertilizer (see treatments labeled 3 and 4) grew aswell or better than the reference plant grown from uninoculated seedwith recommended amounts of dissolved phosphorus fertilizer.

DESCRIPTION OF DRAWINGS

FIG. 1: Endophytic bacteria isolated from plants corn, sorghum,sugarcane: a) corn plants washed in tap water; b) separated roots andshoots; c) separated and chopped corn shoots for further surfacesterilization and grinding to isolate endophytic bacteria

FIG. 2: Endophytic bacteria isolated from corn plants

FIG. 3: Endophytic bacteria isolated from sorghum plants

FIG. 4: Endophytic bacteria isolated from sugarcane plants

FIG. 5: a), b), and c) showing endophytic bacteria isolated from cornplants that solubilize P; based on sperber's PO₄ solubilizing assay

FIG. 6: a), b), and c) showing endophytic bacteria isolated from sorghumplants that solubilize P; based on sperber's PO₄ solubilizing assay

FIG. 7: a), b), and c) showing endophytic bacteria isolated fromsugarcane plants that solubilize P_(i) based on sperber's PO₄solubilizing assay

FIG. 8: a) control plants in complete Hoagland solution; b) corn plantinoculated with T6 grown in modified Hoagland solution amended with 0.2g rock phosphate; c) corn plant inoculated with T6 grown in modifiedHoagland solution amended with 0.1 g iron-phosphate; d) and e)non-inoculated corn growing in phosphate deficient Hoagland solution

FIG. 9: root and shoot length plotted for 1) control plant grown inphosphate deficient Hoagland solution show very poor growth as measuredby their poor root and shoot lengths indicative of yield; 2) corn plantgrown in complete Hoagland solution as expected are healthy and showgood/optimal root and shoot growth; 3) corn plant grown in phosphatedeficient Hoagland solution amended in iron-phosphate nanoparticles showroot length and shoot lengths better than the reference/control plants;4) corn plants grown in phosphate deficient Hoagland solution amended inrock-phosphate also show root length and shoot lengths better than thereference/control plants

FIG. 10: Total chlorophyll content comparing T6, and T4 inoculated corngrown on rock phosphate (T4RP, T6RP) and iron phosphate, respectively(T4IP, T6IP), and control plants (D) and corn plants grown in fullstrength Hoagland solution (C)

FIG. 11: a) sorghum control plants in complete Hoagland solution; b)sorghum plant inoculated with T6 grown in modified Hoagland solutionamended with 0.1 g iron phosphate nanoparticles; c) sorghum plantinoculated with T6 grown in modified Hoagland solution amended with 0.2g rock-phosphate; d) non-inoculated sorghum growing in phosphatedeficient Hoagland solution

FIG. 12: root and shoot length plotted for 1) control plant grown inphosphate deficient Hoagland solution show very poor growth as measuredby their poor root and shoot lengths indicative of yield; 2) sorghumplant grown in complete Hoagland solution as expected are healthy andshow good/optimal root and shoot growth; 3) sorghum plant grown inphosphate deficient Hoagland solution amended with iron-phosphatenanoparticles show root length and shoot lengths same asreference/control plants; 4) sorghum plants grown in phosphate deficientHoagland solution amended with rock-phosphate show root length and shootlengths slightly better than the reference/control plants.

EXAMPLE 1: ISOLATING ENDOPHYTIC BACTERIA FROM FRESH PLANT SAMPLES

Fresh samples of corn, sorghum, and sugarcane plants were acquired andwashed in tap water.

The roots and shoots from each plant were separated and chopped. Theywere then surface sterilized to eliminate any epiphytic bacteria and tofacilitate isolation of only endophytic bacteria. The samples were thenground to isolate endophytic bacteria. Endophytic bacteria isolated fromcorn plants included T4, T6, and C8. Endophytic bacteria isolated fromsorghum plants included J-1, J-2/1, J-2/2, J-3/1, J-3/2, J-3/3, and J-4.Endophytic bacteria isolated from sugarcane plants included S-1/1,S-1/2, S-5, S-7, and S-8.

EXAMPLE 2: TESTING ENDOPHYTIC BACTERIA FOR INORGANIC-P SOLUBILIZATION

Sperber's media for screening Pi solubilizing endophytic bacteria: Thebasal Sperber (1958) medium was used and contained glucose 10.0 g/l,yeast extract 0.5 g/l, CaCl₂) 0.1 g/l, MgSO₄.7H₂O 0.25 g/l and agar 15.0g/l. The medium was supplemented with 2.5 g/L of Ca₃(PO₄)₂(TCP-tricalcium phosphate) as P source to appraise the ability of thestrains to mobilize inorganic P sources. The pH of the medium wasadjusted to 7.2 before autoclaving. The media were distributed in 9 cmdiameter Petri plates and marked in four equal parts aftersolidification. Using the drop plate method, each part was inoculatedwith innocula. All tests were performed with four replications.Inoculated plates were incubated in dark at 27 degree C. and thediameter of clear zone (halo) surrounding the bacterial growth as wellas the diameter of colony were measured after 10, 20 and 30 days.

TABLE 1 Results of Sperber's PO₄ solubilizing Assay Endophytic bacteriaisolated from corn (C, T), surgarcane (S) and Phosphate solubilizingsorghum (J) activity (zone in cm) C-8 Very low (0.1 cm) T-4 Medium (1cm) T-6 Medium (0.6 cm) J-2/2 Very low (0.2 cm) J-3/2 Very low (0.1 cm)J-3/1 Very low (0.3 cm) J-4 Medium (1 cm) S-1/1 Very low (0.1 cm) S-1/2Very low (0.3 cm) S-5 Very high (4 cm) S-7 Very high (5 cm) S-8 High (3cm)

EXAMPLE 3: TESTING ENDOPHYTIC BACTERIA FOR ACID PHOSPHATASE PRODUCTION

Screening for acid phosphatase producing endophytic bacteria: Theisolated strains were grown in 50 ml of liquid medium (0.1% Ca-phytate;1.5% glucose; 0.2% NH₄NO₃; 0.05% KCl; 0.05% MgSO₄.7H₂O; 0.03%MnSO₄.4H₂O; 0.03% FeSO₄.7H₂O, pH 5.5) in 500-ml flask and incubated at28 degree C. for 48 hours on reciprocal shaker (200 rpm). The cells werecollected from 1 ml of culture by centrifugation at 5000×g for 10minutes in cool room (40 C) and re-suspended in acetate buffer (0.2 M,pH 5.5). The reaction mixture was prepared. It consisted of 0.8 mlacetate buffer (0.2 M, pH 5.5) containing 1 mM Na-phytate and 0.2 ml ofcell suspension. After incubation for 30 minutes at 37 degree C., thereaction was stopped by adding 1 ml of trichloroacetic acid. One mlaliquot was analyzed for inorganic phosphate liberated using thecolorimetric procedure. One unit of enzyme activity was defined as theamount of enzyme liberating 1 n mol of inorganic phosphate per minute.

TABLE 2 Results of acid phosphatase assay Endophytic bacteria isolatedfrom corn (C, T), Concentration of acid surgarcane (S) and phosphataseproduced sorghum (J) (mg/l) J-1 0.60 S-5 0.60 C-8 0.58 J-4 0.57 S-7 0.62J-2/2 0.52 T6 0.60 J3/1 0.62 T4 0.61 S-1/2 0.60 J-2/1 0.59

EXAMPLE 4: TESTING ENDOPHYTIC BACTERIA FOR ALKALINE PHOSPHATASEPRODUCTION

Screening for alkaline phosphatase producing endophytic bacteria: Theendophytic bacteria were grown in blood agar for 24 h. One colony wastransferred and incubated at 37 degree C. in 2.75 ml of propanediolbuffer (0.2 mol/liter, pH 7.5) containing 2 mg of5-bromo-4-chloro-3-indolyl phosphate previously dissolved in 0.25 ml ofN,N-dimethyl formamide. 0.2 ml of MgCl₂ (5 mmol/liter) was added as anactivator. Alkaline phosphatase production was examined every 30 minutesfor 4 h by looking for a blue-green indigo precipitate development onthe bacterial growth causing the entire solution to become blue.

TABLE 3 Results of alkaline phosphatase assay Endophytic bacteriaisolated from corn (C, T), surgarcane (S) and Change of color to sorghum(J) indigo blue-green C-8 Negative T-4 Negative T-6 Positive J-1Negative J-2/1 Negative J-2/2 Negative J-3/2 Negative J-3/1 NegativeJ-3/3 Negative J-4 Negative S-1/1 Negative S-1/2 Negative S-5 NegativeS-7 Negative S-8 Negative

EXAMPLE 5: TESTING INOCULATION EFFICACY OF ENDOPHYTIC BACTERIA

The inoculum for endophytic bacteria was grown under controlledconditions for 48 hrs to inoculum density of 10⁸ to 10¹⁰ cfu/ml. Theinoculum was centrifuged and suspended in sterile PBS to a concentrationof 10⁸ cfu/ml. The seeds were surface sterilized with 95% ethanol for 2min and 2.5% sodium hypochlorite for 20-30 min followed by washing seventimes in sterile water. Surface sterilized seeds were soaked in sterilePBS containing endophytic bacteria and placed in a temperaturecontrolled incubator shaker at 25 degree C. for exactly 30 minutes. Theinoculated seeds were washed with 70% alcohol for 2 minutes and with 2%sodium hypochlorite followed by washing with sterile water 5 times. Thesurface sterilized seeds were placed in sterile petriplates containing0.7% of water agar, 5-10 seeds per plate. The seed containing plateswere transferred to growth chamber set at 30 degree C. and left for 48hours to germinate. Well germinated seeds with shoot and roots wereseparated and surface sterilize with 95% of ethanol for 5 min and 20 minwith 4% sodium hypochlorite followed by 4-5 times sterile water rinse.The water rinsed root and shoot parts were transferred to PBS containingsolution and ground to rapture the tissue. 1 ml of ground tissue wasdiluted in 9 ml of sterile water serial dilutions were continued toobtain 100 and 1000 fold dilution and spread on nutrient agar plates.After growth the colonies were counted and tabulated. Non-inoculatedseeds served as negative controls.

TABLE 4 Inoculation efficacy of endophytic bacteria in corn and sorghumcfu/ml (calculated using Sample description 1000 fold dilution)Corn-root inoculated with T6 2.1 × 10⁶ Corn-shoot inoculated with T6 1.7× 10⁵ Sorghum-root inoculated with T6 2.6 × 10⁶ Sorghum-shoot inoculatedwith T6 3.4 × 10⁵ Un-inoculated corn and sorghum seeds showed zeroinoculum density in roots and shoots

EXAMPLE 6: TESTING THE EFFICACY OF ENDOPHYTIC INOCULATION FORELIMINATING CHEMICAL FERTILIZERS IN CONTROLLED HYDROPONIC SYSTEMS

Standard Hoagland solutions (hydroponic nutrient solutions) wereprepared and contained Ca(NO₃)₂.4H₂O, NH₄NO₃, KCl, KNO₃, Mg(NO₃)₂.6H₂O,KH₂PO₄, Fe(NO₃)₃.9H₂O, Na HEDTA, MnCl₂.4H₂O, H₃BO₃, ZnSO₄.7H₂O,CuSO₄.5H₂O, and Na₂MoO₄.2H₂O. The young corn seedlings cannot toleratefull strength Hoagland solution. Hence ½ strength Hoagland solution wasused from VE to V1 vegetative stage. The plants were grown until V3vegetative growth stage because phosphate deficiency symptoms can beobserved during V1 to V3 growth stage. Phosphate deficient Hoaglandsolution was prepared by eliminating KH₂PO₄.

The viable inoculated seeds were placed in a muslin cloth and washedwith running tap water and dried by placing on autoclaved tissue paper.The seeds were surface sterilized in laminar flow hood with 70% alcoholfor 30 minutes and then by washing with 10% sodium hypochlorite solutionfor 20 minutes. After surface sterilization, the seeds were washed withsterile water, excess water removed by blotting with autoclaved tissuepaper, and then air dried under laminar flow. Five to ten surfacesterilized and dried seeds were then placed in sterile petriplatescontaining water soaked blotting paper and transferred to growth chambermaintained at 25-27 degree C. and left for 48 hours to germinate. Afterseeds germinated, they were transferred to hydroponic reactors.Hydroponic systems were maintained under greenhouse conditions at 23-24degree C., 70% relative humidity, and 12 hours photo-period. Seeds werefirst grown in full strength Hoagland solution and then transplantedinto hydroponic reactors for all the treatments and controls. Fivereplications were used for all treatments and controls.

Hydroponic reactors constituted of root permeable plastic buckets. Airpump with air controllers were used to provide aeration to the plants inhydroponic systems and all systems were maintained under greenhouseconditions at a temperature of 24±2 degree C. and relative humidity of70±3%. After control plants showed phosphate deficiency symptoms, theplants were removed from hydroponic reactors and washed under runningtap water to completely remove Hoagland solution. The plant was driedwith blotting paper while taking care to not damage the roots. The rootswere separated from the shoots by using sharp scissors and were weighedto record fresh weight of samples. The root and shoot samples were alsodried in the dry air oven at 40 degree C. for 2 days and their dryweight was recorded. The root:shoot ratio of individual plants wascalculated and recorded.

Reduced leaf area and degradation of chlorophyll in leaves is alsosymptomatic of phosphate deficiency so we also measure chlorophyll a andchlorophyll b in plants. A single leaf per plant was used for obtaining10 leaf discs of 1 cm each and weighed. Five of these leaf discs wereplaced per tube containing 5 ml of 1:1 ratio of DMSO:acetone and thetubes were placed in the dark overnight to allow chlorophyll leaching.After chlorophyll leaching the solution turns green and theconcentration of chlorophyll in leaves is calculated by measuringabsorbance at 645, and 663 nm. The total chlorophyll is estimated usingthe following formulas:Chlorophyll II a (g/l)=0.0127 A ₆₆₃−0.00269 A ₆₄₅Chlorophyll II b (g/l)=0.0029 A ₆₆₃−0.00468 A ₆₄₅Total Chlorophyll (g/l)=0.0202 A ₆₆₃+0.00802 A ₆₄₅

What is claimed is:
 1. A method comprising using a plant bacterialendophyte that is heterologous to the seed for the purpose of reducingphosphorus fertilizer requirement for optimal plant growth and yield inorganic and conventional agriculture compared to a referenceagricultural plant grown under the same conditions with no reduction inphosphorus fertilizer that consists essentially of the followingsteps: 1) growing an endophytic bacteria having the ability to produceorganic acids, acid phosphatase enzyme, and alkaline phosphatase enzymeto a inoculum density of 10⁸ to 10¹⁰ cfu/ml; 2) suspending the saidinoculum in sterile phosphate buffer saline medium to a concentration of10⁸ cfu/ml thereby providing an endophytic inoculum; 3) preparing thecorn, sorghum, wheat, rice, and other vegetable, fruit, flower or grassseeds or plant parts by surface sterilizing with 95% ethanol for 2 minand 2.5% sodium hypochlorite for 20-30 min followed by washing seventimes in sterile water; 4) soaking the aforementioned surface sterileseeds or plant parts in the said endophytic inoculum of step 2; 5)henceforth placing in a temperature controlled incubator at 25 degree C.for exactly 30 minutes with or without gentle shaking at 40-80 rpm, thenwashing the thus prepared inoculated seeds or plant part with 70%alcohol for 2 minutes and with 2% sodium hypochlorite followed bywashing with sterile water 5 times; 6) treating the said prepared seedsor plant parts of step 5 with other seed treatments and coatings; 7)drying the said seed of step 6 before planting; and 8) planting the saidprepared seeds or the said plant part in a plant growth medium with lessthan the recommended phosphorus fertilizer amount wherein thephosphorous is applied as triple superphosphate, diammonium phosphate,rock phosphate, manure or another form.
 2. The method of claim 1,wherein the inoculated plant, plant part or seed is introduced in aplant growth medium in an amount effective to increase the yield ofplants grown in said plant growth medium.
 3. The method of claim 1, inwhich said endophytic inoculum, is introduced into a plant growth mediumas part of an inoculant composition comprising at least 1×10⁵ colonyforming units of said endophytic inoculum per gram or per milliliter ofinoculant composition.
 4. The method of claim 1, in which saidendophytic inoculum, is introduced into a plant growth medium as part ofa treated plant part.
 5. The method of claim 4, in which said treatedplant part comprises at least 1×10⁵ colony forming units of saidendophytic inoculum per kilogram of plant part.
 6. The method of claim1, in which said endophytic inoculum is introduced into a plant growthmedium at a rate of at least 1×10⁵ colony forming units of saidendophytic inoculum per acre of plant growth medium.
 7. A The method ofclaim 1 wherein said endophytic inoculum is applied to plant seeds, oneyear before said plant seed is planted in a plant growth medium.
 8. Amethod comprising using NRRL B 67827 that is heterologous to the seedfor the purpose of reducing phosphorus fertilizer requirement foroptimal plant growth and yield in organic and conventional agriculturecompared to a reference agricultural plant grown under the sameconditions with no reduction in phosphorus fertilizer that includes thefollowing steps: 1) growing NRRL B 67827 having the ability to produceorganic acids, acid phosphatase enzyme, and alkaline phosphatase enzymeto a specific inoculum density of 10⁸ to 10¹⁰ cfu/ml; 2) suspending thesaid inoculum in sterile phosphate buffer saline medium to aconcentration of 10⁸ cfu/ml thereby providing an endophytic inoculum; 3)preparing the vegetable, fruit, flower, or grass seed, or plant parts bysurface sterilizing with 95% ethanol for 2 min and 2.5% sodiumhypochlorite for 20-30 min followed by washing seven times in sterilewater; 4) soaking the aforementioned seed or plant parts in the saidendophytic inoculum; and 5) henceforth placing in a temperaturecontrolled incubator at 25 degree C. or any other temperature dependenton seed type or plant parts with or without gentle shaking at 40-80 rpmfor exactly 30 minutes, then washing the thus prepared inoculated seedsor plant parts with 70% alcohol for 2 minutes and with 2% sodiumhypochlorite followed by washing with sterile water 5 times; 6) treatingthe said prepared seeds or plant parts of step 5 with other seedtreatments and coatings; 7) drying the said seed of step 6 beforeplanting; and 8) planting the said prepared seeds or the said plant partin a plant growth medium with less than the recommended phosphorusfertilizer amount wherein the phosphorous is applied as triplesuperphosphate, diammonium phosphate, rock phosphate, manure or anotherform.
 9. The method of claim 1 in which said endophytic inoculum whenapplied to the said plant seed or plant growth medium can reduce oreliminate leaching of the applied phosphorus fertilizer in conventionaland organic agriculture.
 10. The method of claim 8 wherein NRRL B 67827when applied to the said plant seed or plant growth medium can reduce oreliminate leaching of the applied phosphorus fertilizer in conventionaland organic agriculture.
 11. The method of claim 8 wherein theinoculated plant, plant part or seed is introduced in a plant growthmedium to increase yield with optimal growth.
 12. The method of claim 8,in which the said strain NRRL B 67827 is introduced into a plant growthmedium as part of an inoculant composition comprising at least 1×10⁵colony forming units of NRRL B 67827 per gram or per milliliter ofinoculant composition.
 13. The method of claim 8, in which the saidstrain NRRL B 67827 is applied to the said plant seed one year beforethe said plant seed is planted in a plant growth medium.